Cutaneous T cell lymphoma (CTCL) is a disfiguring, incurable cancer. For patients with advanced disease, current therapies are inadequate, and outcome is poor. An incomplete understanding of CTCL molecular regulators has limited development of effective targeted therapies. One candidate regulator is p38?, the gene expression of which is selectively increased in CTCL cell lines and patient samples, but not healthy T cells. We demonstrate that inhibition or silencing of p38? inhibits proliferation and induces CTCL cell death. The NF-?B pathway is constitutively active in CTCL, provides a complementary T cell signaling pathway to p38?, and can be inhibited by histone deacetylase inhibitors (HDACi). HDACi, which are currently the most effective clinically approved cytotoxic compounds against CTCL, demonstrate synergistic killing when combined with p38? Inhibition. Our objective is to understand and exploit the p38? pathway in CTCL, using a combination of molecular, chemical, and genetic approaches. Our first Aim is to determine the mechanisms by which p38? inhibition induces cell death in CTCL. We will define the kinase cascade involved in p38? inhibition-induced CTCL cell killing and identify phosphorylation targets of p38? signaling; use a synthetic lethal RNAi screen to identify signaling components that cause cell death upon depletion in the presence of p38? inhibition; and determine the extent to which combined inhibition of p38? and complementary pathways, including HDACs, induce synergistic therapeutic effects. We will validate identified proteins for the ability to affect downstream signaling and cellular responses in vitro and in vivo, using CTCL cell line xenograft and patient-derived xenograft (PDX) models. Our second Aim is to develop novel p38? inhibitors for potential therapeutic application. Using high throughput screening and molecular modeling, we identified the multi-kinase inhibitor F7 (also known as PIK75), and showed it is an ATP-competitive p38? inhibitor with nanomolar cytotoxic efficacy against CTCL cells. To develop a more selective p38? inhibitor, we will combine ligand- and structure-based computational methods with organic synthesis; using F7 as a scaffold molecule, we will identify F7 analogs and derivatize F7 to have higher a binding affinity for p38? than other kinases. In addition, we will use CRISPR-based screening to identify novel functional domains and non-conserved sites for developing allosteric next-generation therapeutics. We will synthesize the various analogs and validate hits for CTCL cytotoxicity and p38?-specific kinase inhibition in vitro and in vivo, using CTCL xenograft and PDX models. We expect that successful completion of this proposal will yield mechanistic information about the unique biological and clinical relevance of p38? signaling and complementary pathways in CTCL. Importantly, validation of a specific p38? inhibitor with efficacy in CTCL animal models will have immediate relevance for CTCL therapy.